Process for fermenting carbohydrates to produce butanol and isopropanol



Patented May 27, 1947 PROCESS FOR FERMENTING CARBOHY- DRATES TO PRODUCE BUTANOL AND ISO- PROPANOL Samuel C. Beesch and David A. Legg, Philadelphia, Pa., assignor to Puhlicker Industries Inc., a corporation of Pennsylvania No Drawing. Application July 27, 194

Serial No. 546,904

9 Claims.

The present invention relates to bacterial fermentations and it relates more particularly to the production of butyl alcohol, isopropyl alcohol and other substances by the bacterial fermentation of carbohydrate mashes.

An object of the present invention is to provide a new and improved process for the production of butyl and isopropyl alcohols by bacterial fermentation. Another object of the present invention is to provide for fermentation of carbohydrate mashes by a certain newly discovered organism, to be hereinafter described, which gives substantial yields of butyl and isopropyl alcohols. Still another object of the present invention is to produce butyl and isopropyl alcohols by means of a certain newly discovered organism which is capable of producing substantial yields of said alcohols not only from sugars but also from starches without the need for preliminary hydrolysis thereof.

Other objects and advantages of the present invention will be apparent in the following detailed description and appended claims.

It is well known in the art that sugar solutions or mashes can be fermented by various bacteria to produce butyl and isopropyl alcohols. These organisms heretofore employed have, however, been incapable of fermenting starches; it having been necessary first to hydrolyze the starch to sugar before the fermentation could be carried out.

We have discovered and isolated a new organism which we have named Clostridium amylosaccharo butyl-propylicum which is capable of fermenting not only sugars but also starches without preliminary hydrolysis to give substantiul yields of butyl and isopropyl alcohols plus relatively small percentages of acetone. This organism may be isolated from soil by making heatshocked enrichment cultures in molasses medium,

plating anaerobically, selecting bacterial coloniesof the type to be hereinafter described, and testing their fermentative capacity on molasses and starch media of the kind set forth in the examples given hereinbelow.

The organism will be described using the descriptive chart of the Society of American Bacteriologists for facility of identification.

TABLE Name: Clostridium amylo-saccharo butylpropylicum.

Source: Soil.

I. Morphology 1. Vegetative cells.

Medium usedPotato glucose medium (300 gms. potato (Idaho) moist weight, 10 gms. of glucose, 1 gm. ammonium sulfate, 3 gms. calciumcarbonate per liter).

Incubation 20 hrs. at 30-"C.

Stain used-Nigrosin without heat.

FormShort and long rods.

Arrangement-Single and chains.

Usual limits of length-25 to 12.0 a; of diameter0.60 to 2.8 ,u.

Size of majority-51 by 1.3 a.

Ends-Rounded.

2. SporangiaPresent.

Medium used-Potato glucose medium.

Incubation36 hrs. at-30 C.

Stain used-Nigrosin without heat.

FormSpindled and clavate.

3. EndosporesPresent.

Medium usedPotato glucose medium.

Incubation-'72 hrs. at 30 C. 1

Stain usedNigrosin without heat.

Location of endospores-Sub-terminal to terminal. 7

FormCylindrical to oval.

Usual limits of length.0.8 to 2.8 n; of diameter-0.5 to 2.0 l

Size of majority1.8 by 1.0 ,u.

4. Motility.

Medium used-Glucose broth (5 gms. peptone, 3 gms. beef extract, 10 gms. glucose per liter).

Incubation20 hrs. at 30 C.

MotilityMoti1e.

Medium used-Nutrient agar (agar 17 gms., glucose 20 gms.,molasses 8 gms., peptone 6 gms.;beef extract 3 gms., (-NH4)2SO-4 1 gm. per liter).

Incubation-.40 hrs. at 30 C.

Motility-Motile.

5. Flagella-Present.

Medium used-Molasses mash (40' gms. sugar calculated on Cuban invert molasses, ammonium sulfate 2.2 gms., calcium carbonate 2.4 g., calcium acid phosphate 1.3 gms. per liter). 0

Incubation-24 hrs. at 30 C.

Stain-used-Lofllers flagella stain.

AttachmentPeri-trichous.

6. Irregular forms-Present.

Medium used-Potato glucose medium. Incubation72 hrs. at 30 C.

3 4 '7. Staining reactions. 4. Production of indole.

(a) Gram stain. Medium used-Glucose tryptophane (gin- Medium usedPotato glucose medicose 2.5 gms., tryptophane 1.0 gm. per litre).

Incubation-20 hrs. at 30 C. 5 Incubation96 hrs. at 36 C.

Stain used-Kopeloff Beerman modi- Test usedp-Dimethylaminobenzaldehyde.

fication. Indole-Absent.

Stain-Positive; variable after 24 hrs. 5. Production of hydr s phide.

(b) I din t i Medium used-Lead acetate agar (agar 15 Medium used-Nutrient agar. m B tot ypt 0 ms., l e 10 Incubation-48 hrs. at 30 C. lead acetate 0.2 gms. P r lite Granulose-Positive, Incubation-72 hrs. at 30 C.

Hydrogen sulphide-Absent or present in 11. Cultural characteristics traces.

15 6. Relation to oxygen. 1. Agar colonies. (a) Medium used-Nutrient agar.

Medium used-Nutrient agar. Incubation48 hrs. at 30 C.

Incubation-48 hrs. at 30 C. Growth (aerobic incubation) Absent.

FormRound, or circular, tendency to Growth (anaerobic incubation)-Abunspread out. dant.

Surface-Pearly lustre, smooth. (1)) Medium used-Potato glucose medium EdgeEntire. in deep tubs. Elevation-Convex. Incubation24 hrs. at 30 C. Optical character-Opalescent with dark Growth (aerobic incubati0n)-Abuncenters finely granular structure, light 25 dant. tan. Growth (anaerobic incubation)-Abun- 2. Agar stroke. dant.

Medium used-Nutrient agar. 7. Litmus milk.

Incubation-96 hrs. at C. Incubation30 C.

Growth-Anaerobic, abundant. 30 Reaction (3 days)Acid.

FormSpreading. Curd (10 days) Acid curd. Lustre-Glistening. Peptonization (l5 days)-Slight if any.

Chrom0 n s s ht ea to an. Reduction of litmus (1 day) Reduced.

0dorButylic. 8. Nitrate reduction.

Consistency-Riscid. Medium-8% potato, 1% glucose, 0.1%

Change in color of mediumNone. KNO:.

3. Nutrient broth. Incubation-1 day to 4 days.

1-, t broth 5 peptone Test useda-Naphthylamine sulfanilic acid.

' 3 gms. beef exract per liter) ReductiQn Nne;

Incubation 30o Q 40 9. Fermentation reactions. surface growth None (a) Acid and gas product on. clouding slight. Medium usedNutr1ent broth+10 gm. odor None. per liter of carbohydrate or alcohol to be tested.

4. Gelatin stab.

Medium used-Nutrient gelatin (gelatin 150 gms., glucose 10 gms., peptone 5 gms., beef Acid pm Gas proextract 3 gms. per liter). Oamhydme 51mm duction g fi gf Incubation-4 days 30 C. GrowthBest below surface to bottom line 0 Eseulin Puncture 'iiitiltiii j i Liquefaction-30 days none. Rhamnose- Change in color of medium-None. i gggg E i 5. Potato stroke. u I Galaetose" Medium-Stenhzed potato. 55 Incubation96 hrs. at 30 C. gg---- i i Growth-Abundant. Maltese I: FormSpreading. gz gg i I I Luster-Glistening. i i Chromogenesis-Light cream to yellow. Odor-Butylic. i Consistency-Viscid. Change in medium-None to slight liquefaction.

III. Physzologzcal charactemstzcs OS} 1. Temperature relations: Optimum fermentation Mehbmse temperature-29-32 C. 2. Relation to reaction of medium: Optimum Negauve posmve' fi l p An important characteristic of these bacteria 3. Chromogenesis. from a commercial standpoint is their ability to Nutrient agarPearly lustre with tan color. produce consistent yields of solvents ranging be- Nutrient gelatinNone. tween about 30% to about 33% or more from in- Potato-Light cream to yellow. vert sugar, glucose, or mixtures of these sugars with sucrose in mashes wherein the percentage of sucrose does not exceed about 30% of the total sugar. When the organism is used to ferment blackstrap molasses, which contians sucrose and invert sugar in the ratio of about 60 to 40, the yields of solvents are somewhat lower unless part of the sucrose is inverted.

Another important characteristic of these bacteria is their ability to produce substantial yields of solvents from unsaccharified cereal mash to which suitable nutrients have been added.

Still another important characteristic of these bacteria is their ability to ferment mashes wherein the sugar concentration is as high as 7% or more without any appreciable falling 011: in the yields of solvent.

The following are illustrative examples of fermentations carried out with Clostridium amylosaccharo butyl-propylicum:

EXAMPLE 1 A high-test molasses containing about 20% sucrose and about 56% invert sugar was used to form an aqueous mash having a total sugar concentration of about 6%. To about 3,000 parts of this mash were added about parts of ammonium sulphate, 12 parts of calcium carbonate and /2 part of P205 as calcium superphosphate. The mash was then sterilized by heating for about 30 minutes at about pounds pressure. The steril- EXAMPLE 2 The procedure of Example 1 was repeated using a mash having about 7% total sugar.

The fermentation was complete in about 60 hours and analysis showed 20.3 grams of solvent per liter of mash, which corresponds to a yield of 30.5% based on the total sugar content of the mash.

EXAMPLE 3 A blackstrap molasses containing about 35.7% sucrose and 17% reducing sugars was used to form an aqueous mash having a total sugar concentration of about 6%. To about 3,000 parts of this mash were added about 10 parts of ammonium sulphate and 12 parts of calcium carbonate. The mash was then sterilized, cooled, and inoculated with about 3% of a 24 hour active culture of Clostridium amylo-saccharo butyl-propylicum.

The fermentation was complete in about 60 hours and, on distillation, yielded 15.35 grams per liter of solvents corresponding to a yield of 25.6% based on total sugar content.

EXAMPLE 4 About 3,000 parts of mash were made up to contain about 183 parts of dry corn, 9 parts of ammonium sulphate, and 12 parts of calcium carbonate. The mash was cooked for about 2 /2 hours at about 15 pounds pressure, after which it was cooled to about 30 C. and inoculated with about 3% of a 24 hour active molasses culture of Clostridium amylo-saccharo butyl-propylicum.

Upon completion of the fermentation, distillation yielded 14.6 grams per liter of total solvents corresponding to a yield of 24% of the dry corn or approximately 33% of the starch.

The carbohydrate concentration of the final mash may be as high as 7% or even more. However, if the sugar or grain source is deficient in suitable degraded protein, it is desirable to add soluble nitrogen in the form of ammonia or its salts, or organic derivatives including urea, com steep water or other sources of degraded protein, etc. Furthermore, if the raw material is deficient in bufiering materials, steps must be taken to keep the pH of the mash from falling below 4.8 during the fermentation. This can be done by gradually adding alkaline neutralizing agents, such as alkali or alkaline earth oxides, hydroxides or carbonates, or alkaline ammonium compounds, in suitable amounts and at suitable rates. The amounts so added should not raise the pH appreciably above 6.8 nor should it be more than,

is necessary to prevent the pH from falling below 4.8. The amounts required can readily be determined by experiment for each type of raw material and neutralizing agent.

The average composition of the solvents produced when a sugar mash, such as those of Examples 1, 2 and 3, is fermented is about 65-72% butyl alcohol, 26-32% isopropyl alcohol and 24% acetone, although a trace of ethyl alcohol may also be found. Some variation in the composition of the solvents may result, however, due to change in the composition of the medium or to the method of maintaining the pH above 4.8.

The organism may be preserved indefinitely in the spore form by pouring molasses or potato medium culture containing clostridia or spores on sterile soil and drying under aseptic conditions.

In order to start an active culture, we may inoculate, for example, 10 to 20 cc. of sterile 10% potato medium with about 0.05 gram of the soil culture, heat shock if desired, and incubate at about 30 C. for about 24-36 hours. About 3% of this culture may then be used to inoculate a sterile molasses medium containing 4-6% of sugar, about 0.2% of ammonium sulphate, and 0.3% of calcium carbonate. After 16-24 hours incubation at 30 0. this culture may be used to seed other mashes of similar composition, in each case using about 2-4% of inoculum. At least 8 transfers can be made in this way so that any desired quantity of inoculum can be developed by daily transfers to culture vessels of increasing size.

About 2-4% of the inoculum is used in the fermentation. The inoculum may contain, in addition to the sugary medium, assimilable nitrogen and other nutrients as required and may be adjusted to a pH of about 4.8 to 6.8.

The fermentation may be carried out at between about 28 and C. for about to 72 hours.

The organism described hereinabove is capable of fermenting other sugary mashes, as for example, mashes made from beet sugar or beet molasses; cane sugar; hydrolyzed carbohydrates, such as dextrine, inulin and syrups; and other industrial sources of sugar, with the limitation noted above, namely, that the sugar should not contain more than about 30% sucrose for optimum results.

The organism is capable of fermenting other cereal grains, in addition to corn; as for example, wheat, oats, rye, barley, etc.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it is therefore desired that the present embodiments be considered in all respects as illustrative and not restrictive, reference being had to the appended claims rather than to the foregoing description to indicate the scope of the invention.

Having thus described our invention, what we claim as new and desire to protect by Letters Patent is:

1. A process for producing butyl and isopropyl alcohols which comprises forming an aqueous mash containing a carbohydrate, of the group consisting of cereals and sugars and containing assimilable nitrogen, inoculating the mash with a bacterial culture containing Clostridium amylosaccharo butyl-propylicum, and incubating said inoculated mash at a temperature and for a time adequate to permit fermentation thereof.

2. A process for producing butyl. and isopropyl alcohols which comprises forming an aqueous mash containing not more than about 7% of a carbohydrate of the group consisting of cereals and sugars and containing assimilable nitrogen, inoculating the mash with a bacterial culture containing Clostridium amylo-saccharo butylpropylicum and incubating said inoculated mash at between 28 and 35 C. for about 45 to 72 hours while maintaining its pH between about 4.8 and 6.8, thereby to permit fermentation of said mash by said bacterial culture.

3. A process for producing butyl and isopropyl alcohols which comprises inoculating an aqueous mash of a sugary material with a bacterial culture containing Clostridium amylo-saccharo butyl-propylicum and maintaining the mash at a temperature suflicient to bring about active fermentation by the action of said bacterial culture.

4. A process for producing butyl and isopropyl alcohols which comprises forming an aqueous mash containin not more than about 7% of a sugary material and containing assimilable nitrogen, inoculating the mash with a bacterial culture containing Clostridium amylo-saccharo butyl-propylicum and incubating said inoculated mash at a temperature and for a time adequate to permit fermentation thereof.

5. A process for producing butyl and isopropyl alcohols which comprises forming an aqueous mash containing a sugary material and suitable bacterial nutrients, inoculating said mash with a small amount of an active bacterial culture containing Clostridium amylo-saccharo butylpropylicum, and incubating the inoculated mash at a temperature adequate to bring about active fermentation of said mash, for a time sufiicient to convert substantially all the sugary material to products of fermentation.

6. A process for producing butyl and isopropyl alcohols which comprises forming an aqueous mash containing a cereal starch carbohydrate and containing assimilable nitrogen, inoculating the mash with a bacterial culture containing Clostridium amylo-saccharo butyl-propylicum and incubating said inoculated mash at a temperature and for a time adequate to permit fermentation thereof.

7. A process for producing butyl and isopropyl alcohols which comprises forming an aqueous mash containing a cereal starch carbohydrate and containing assimilable nitrogen, inoculating the mash with a bacterial culture containing Clostridium amylo-saccharo butyl-propylicum and incubating said inoculated mash at a temperature and for a time adequate to permit fermentation thereof while maintaining the pH of the mash between about 4.8 and 6.8.

8. A process for producing butyl and isopropyl alcohols which comprises forming an aqueous mash containing not more than about 7% of a cereal starch carbohydrate and containing assimilable nitrogen, inoculating the mash with a bacterial culture containing Clostridium amylosaccharo butyl-propylicum and incubating said inoculated mash at between 28 and 35 C. for about 45 to '72 hours while maintaining its pH between about 4.8 and 6.8, thereby to permit fermentation of said mash by said bacterial culture.

9. A process for producin butyl and isopropyl alcohols which comprises forming an aqueous mash containing a cereal starch carbohydrate and suitable bacterial nutrients, inoculating said mash with a small amount of an active bacterial culture containing Clostridium amylo-saccharo butyl-propylicum and incubating the inoculated mash at a temperature adequate to bring about active fermentation of said mash, for a time sufficient to convert substantially all the cereal starch carbohydrate to products of fermentation.

SAMUEL C. BEESCH. DAVID A. LEGG.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 2,001,930 Daranyi May 21, 1935 2,063,448 Legg Dec. 8, 1936 2,219,426 Loughlin Oct. 29, 1940 1,725,083 Izsak Aug. 20, 1929 FOREIGN PATENTS Number Country Date 415,312 Great Britain 1934 

